The objective of this work is to study the phenomenon of X-chromosome inactivation or gene dosage compensation using malignant teratocarcinoma stem cells. Our previous studies indicated that certain pseudo-diploid XX-teratocarcinoma stem cell lines contain two genetically active X-chromosomes as long as they are maintained in the undifferentiated state, and that the cells can undergo X-inactivation when they are allowed to differentiate in vitro. We propose here to further explore the extent to which such tumor stem cells can provide a valid model system for the study of X-inactivation. To do this, we will isolate new teratocarcinoma stem cell lines by a variety of techniques, including interspecies microcell fusion, to obtain cells that are heterozygous for electrophoretic variants of X-linked enzymes. The availablilty of such cell lines will enable us to confirm by enzyme gel electrophoresis our earlier conclusions. In other experiments we will study the cytogenetic properties of the X-chromosomes in the cells to determine the temporal relationship between the onset of cytological manifestations of X-inactivation (e.g. late replication of DNA) and of biochemical inactivation. In addition, we will determine whether an inactive X-chromosome can be reactivated by placing it in a teratocarcinoma stem cell. Also, when appropiate interspecies hybrid cell lines are available we plan to carry out blastocyst injections in order to obtain chimeric mice. If the injected cells form functional germ cells in these mice we will have thus created a new line of mice that cary an X-chromosome that expresses X-linked gene products with electrophoretic mobilities different from those which are common in laboratory mice. Such mice will be an invaluable tool in studies of X-chromosome activity during normal embryonic development. The results of the studies described here should lead to a better understanding of the X-inactivation process during normal early embryogenesis and may help to explain gene regulation throughout normal development.